Abstract (English)

After administration of the hematopoietic stem cells inoculum into the eligible patients in the allogeneic setting, many hematopoietic cells reconstitute in the following months to years after transplantation. The reconstituted cells can be pathogen-specific (e.g. cytomegalovirus (CMV)-reactive T cells), leukemia-specific (i.e. graft versus leukemia (GvL) effect) or recipient-specific (e.g. ...

Abstract (English)

After administration of the hematopoietic stem cells inoculum into the eligible patients in the allogeneic setting, many hematopoietic cells reconstitute in the following months to years after transplantation. The reconstituted cells can be pathogen-specific (e.g. cytomegalovirus (CMV)-reactive T cells), leukemia-specific (i.e. graft versus leukemia (GvL) effect) or recipient-specific (e.g. graft-versus-host disease (GvHD)). Thus, valid in vitro immune monitoring strategies should broaden the understanding of CMV immunity, GvL and GvHD, which seems essential for further improvements in allogeneic stem cell transplantation (SCT). Furthermore, approaches of this type should help to dissect allo-specific from tumor-specific immune responses and will help to clarify, how both mechanisms are interconnected and how they can be best put in action for therapeutic purposes. The CMV-specific T cells reconstitution was monitored efficiently, in allogeneic transplanted patients, by classical approaches such as tetramer (TM) staining, intracellular cytokine (ICC) flow cytometry and enzyme-linked immunospot (ELISPOT) assay, in addition to the newly introduced real time-polymerase chain reaction (RT-PCR) assay. In general, the RT-PCR assay was highly sensitive in comparison to the other methods, and was also correlated to all of them. Interestingly, the use of the pp65 protein as CMV antigen enhanced the monitoring capabilities in comparison to the pp65 HLA-A2-restricted peptide, and allowed testing of HLA-A2 negative patients. As an example for assessment of the GvL effect in allogeneic transplanted patients, the Wilms' tumor suppressor gene (WT1)-specific T cells were monitored. Unfortunately the WT1-specific T cells were not detectable by a sensitive ELISPOT assay, but on the other hand and for the first time the established RT-PCR assay was able to track two types of WT1-reactive T cell clones in the same samples. In addition and as not described before, the RT-PCR assay was able to monitor the reconstitution of WT1-specific T cells in patients who were suffering AML and ALL before transplantation. As the minor histocompatibility antigens (mHAgs) play a dual role in the GvL and GvHD, the reconstitution of male-specific mHags (HY)-specific T cells were monitored. The adopted sensitive ELISPOT assay did not detect any HY-specific T cells in all of the tested patients� samples. On the contrary, the established RT-PCR assay successfully detected the HY-specific T cells in one patient who later developed eosinophilia as a GvHD complication. As for the first time these cells were detectable by RT-PCR assay in the allogeneic transplanted patients� peripheral blood, this give a novel concept of predicting GvHD due to the generation of mHAg-specific T cells in the patients body. Due to the still insufficient knowledge on mechanisms involved in GvHD, it was considered a major aim to tailor the allo-reactive T cells in the patients� peripheral blood in order to avoid fatal courses of GvHD. An allogeneic mixed lymphocyte reaction (MLR) model was established and optimized to simulate the presence of the allo-reactive T cells in the GvHD patients� blood. After applying the established MLR model on some patients� samples, weak but significant allo-reactive T cells could be monitored in few samples by using the RT-PCR assay but not ICC flow cytometry. For better detection of the allo-reactive T cells in the patients� peripheral blood, the use of RT-PCR assay is strongly advised for monitoring GvHD. Finally more patients and other cytokines are suggested to be further monitored to give better view. Overall, the results of this work strongly support that monitoring methods can be established to be used in clinical practice in the near future.